The 3′ Untranslated Region of a Plant Viral RNA Directs Efficient Cap-Independent Translation in Plant and Mammalian Systems

Figures

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  Figure 1. Translation in plant systems. (A) Genome organization of thin paspalum asymptomatic virus (TPAV) as a representative of the Tombusviridae, and luciferase reporter construct. Secondary structure and position of the panicum mosaic virus-like CITE (PTE), which binds the eIF4E subunit of eIF4F (arrow) is shown in the 3′ untranslated region (UTR). Curved dashed line indicates potential pseudoknot base pairing between C domain (CCY, Y = pyrimidine), and G domain (GGG). TPAV open reading frames (ORFs), indicated by boxes are named as in Ref. [41]. For each viral luciferase construct tested in panels B–D, the complete 5′ and 3′ UTR of each virus flank the firefly luciferase ORF (cap-independent translation element (CITE constructs). (B) Relative luciferase activity produced in wheat germ extract or tobacco BY2 cell extract by translation of 20 nM uncapped reporter mRNAs (CITE constructs, panel A) containing the 5′ and 3′ UTR of the indicated virus. m2 indicates CITE construct with a CC→AA mutation in the C-domain of the PTE. (C) Translation of 2 nM uncapped pea enation mosaic virus 2 (PEMV2) CITE construct (open bars) or capped, non-viral (CAluc, black bars) mRNA in wheat germ extract in…

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  Figure 2. PTE-mediated cap-independent translation in mammalian systems. (A) Overlay of wheat (blue) and human (amber) eIF4E structures (Swiss PDB Viewer and PovRay, accession numbers 2IDV and 1IPC, respectively), showing tryptophan residues that sandwich around the m7GTP cap. (B) Cis-translation results of uncapped viral reporter RNAs (CITE constructs, Figure 1A) in rabbit reticulocyte lysate (RRL), with relative light units (RLU) shown relative to those produced by the CarMV CITE construct. BlucB is a luciferase reporter containing the 5′ and 3′ UTRs of barley yellow dwarf virus (BYDV), which harbors a BYDV-like translation element (BTE) rather than a PTE in its 3′ UTR. (C) Trans-inhibition of translation of uncapped PEMV2 CITE construct mRNAs by PTEs as in Figure 1C, but in RRL. (D) Cis-translation of uncapped viral CITE construct RNAs in HeLa cell lysate. RLUs are relative to those obtained from firefly luciferase RNA containing the EMCV IRES in its 5′ UTR. (E) Cis-translation results of viral CITE construct RNAs relative to EMCV reporter in DNA-transfected BHK-T7 cells. Plasmids were linearized with Sma I as for in vitro transcription of RNAs used in in vitro assays. The data shown…

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  Figure 3. Electrophoretic mobility shift assay (EMSA) of eIF4E binding to the PTE. Twenty femtomoles of 32P-labeled capped or uncapped PTE RNAs and human (A) or wheat (B) eIF4Es at the indicated concentrations were used in each reaction. Panel (C) shows secondary structures and m2 mutations of the TPAV and PEMV2 PTEs. See Materials and Methods for details.

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  Figure 4. (A) TPAV and TPAVm2 PTE modification and cleavage structure probing patterns generated using the SHAPE reagent benzoyl cyanide (BzCN) in the absence of protein (+ lane) or presence of indicated proteins (lanes 5–11). A and G are dideoxy sequencing lanes, with positions of selected bases indicated at left. Unmodified RNA (− lane) shows background RNA hydrolysis. Gray and white bars to the right show regions with increased or decreased, respectively, modification by BzCN in the presence of eIF4E. Filled circles at right denote regions of autohydrolysis in the absence of BzCN. (B) Positions of human eIF4E-induced changes in SHAPE sensitivity mapped onto the TPAV PTE secondary structure. Double-headed arrow indicates predicted pseudoknot base pairing between the C and G domains, which is disrupted by the CC to AA changes in mutant m2 (boxed in gray).

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  Figure 5. Effect of G4000 mutations in TPAV PTE on structure and translation efficiency. (A) BzCN footprinting of the TPAV PTE with indicated mutation at the hypermodified G4000 residue in the presence of human eIF4E (H), wheat eIF4E (W) or bovine serum albumin (BSA). All proteins were added to final concentration of 2 μM. A and G are dideoxy sequencing lanes showing positions of A and G residues, respectively, in the RNA template. Unmodified RNA (−) shows background RNA hydrolysis, (+) indicates BzN modification in the absence of added protein. Gray bars to the right show regions protected by wheat eIF4E from BzCN modification (G4000U mutant only). Small filled circles at right denote regions of autohydrolysis in the absence of BzCN. (B) TPAV secondary structure showing hypermodified G4000 mutants and sequences protected by wheat eIF4E in the G4000U point mutant from chemical modification by BzCN. (C) Relative luciferase activity produced in wheat germ extract (WGE) and rabbit reticulocyte lysate (RRL) by the translation of indicated mutant TPAV viral CITE construct reporter mRNAs.